Highly efficient expression of human extracellular superoxide dismutase(rhEcSOD)with ultraviolet-B-induced damage-resistance activity in transgenic silkworm cocoons

Extracellular superoxide dismutase(EcSOD)protects tissues from oxidative stress,and thus is considered as a therapeutic agent for many diseases such as atherosclerosis,hypertension,and cancer.However,cost-effective production of bioactive recombinant human EcSOD(rhEcSOD)remains a challenge.Herein,we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms.rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21 mg/g.Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50%and a yield of 3.5±0.5 mg/g.Additionally,N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified.The purified rhEcSOD gained the potent enzymatic activity of 4162±293 U/mg after Cu/Zn ions incorporation.More importantly,rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway.These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.

Reference genes identified in the silkworm Bombyx moil during metamorphism based on oligonucleotide microarray and confirmed by qRT-PCR

Gene expression quantification at mRNA level is very important for postgenomic studies, as gene expression level is the reflection of the special biological function of the target gene. Methods used for gene expression quantification, such as microarray or quantitative real-time polymerase chain reaction （qRT-PCR）, require stable expressed reference genes. Thus, finding suitable control genes is essential for gene quantification. In this study, a genome-wide survey of reference genes during metamorphism was performed on silkworm Bombyx mori. Twelve genes were chosen as putative reference genes based on a whole genome oligonucleotide microarray normalized by external controls. Then, qRT- PCR was employed for further validation and selection of potential reference gene candidates. The results were analyzed, and stable genes were selected using geNorm 3.4 and NormFinder software. Finally, considering factors from every aspect, translation initiation factor 4A, translation initiation factor 3 subunit 4, and translation initiation factor 3 subunit 5 （represented by sw22934, sw14876, and sw13956） were selected as reliable internal controls across the examined developmental stages, while cytoplasmic actin （sw22671）, the commonly used reference gene in a previous study was shown to vary drastically throughout the examined developmental stages. For future research, we recommend the use of the geometric mean of those three stable reference genes as an accurate normalization factor for data normalization of different developmental stages during metamorphism.

Protease inhibitors in Bombyx mori silk might participate in protecting the pupating larva from microbial infection

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like （TIL）-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.

A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori

Serine proteases play important roles in digestion and immune responses during insect development. In the present study, the serine protease gene BmSP36, which encodes a 292-residue protein, was cloned from the midgut cells ofBombyx mori. BmSP36 contains an intact catalytic triad （H57, D102 and S195） and a conserved substrate-binding site （G189, H216 and G226）, suggesting that it is a serine protease with chymotrypsin- like specificity. The temporal and spatial expression patterns of BmSP36 indicated that its messenger RNA and protein expression mainly occurred in the midgut at the feeding stages. Western blotting, immunofluorescence and liquid chromatography-tandem mass spectrometry analyses revealed secretion of BmSP36 protein from epithelial cells into the midgut lumen. The transcriptional and translational expression of BmSP36 was down- regulated after starvation but up-regulated after refeeding. Moreover, expression of the BmSP36 gene could be up-regulated by a juvenile hormone analogue. These results enable us to better define the potential role of BmSP36 in dietary protein digestion at the feeding stages during larval development.

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time-consuming to maintain non-diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HC1 treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incu- bating developing mother eggs from Chinese lineage diapause silkworm strains at 15℃ （15℃-IME）, we were able to prevent the diapause of their daughter eggs; a similar strategy （15℃-IMES） for the germline transformation of the same strains was that the mother eggs were incubated at 15℃, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non-diapause eggs. By combining tempera- ture and light controls, the improved 15℃-IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15℃-IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.

Zinc finger protein rotund deficiency affects development of the thoracic leg in Bombyx mori

The insect limb develops from the imaginal disc or larval leg during metamor- phosis. The molecular mechanisms involved in the development from the larval to the adult leg are poorly understood. Herein, we cloned the full length of a zinc finger gene rotund from Bombyx mori （Bmrn）, which contained a 1419 bp open reading frame, and encoded a 473 amino acid protein. Reverse transcription polymerase chain reaction and Western blot analyses demonstrated that Bmrn was expressed at higher levels in the epidermis than in other tissues tested, and it showed a very high expression level during metamorphosis. Knock-down of Bmrn produced defects in the tarsus and pretarsus, including the fusion and reduction of tarsomeres, and the developmental arrest of pretarsus. Our data showed that Bmrn is involved in the formation of the tarsus and pretarsus, whereas its homologous gene in Drosophila has been shown to affect three tarsal segments （t2-t4）, suggesting that the remodeling of the leg has involved changes in the patterning of gene regulation during evolution.

Characterization of Argonaute family members in the silkworm, Bombyx mori

The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgol and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5＇ end of the small RNA guide strand and of the conserved （aspartate, aspartate and histidine [DDH]） motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.

Genome editing in Bombyx mori: New opportunities for silkworm functional genomics and the sericulture industry

In recent years, research in life sciences has been remarkably revolutionized owing to the establishment, development and application of genome editing technologies. Genome editing has not only accelerated fundamental research but has also shown promising applications in agricultural breeding and therapy. In particular, the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology has become an indispensable tool in molecular biology owing to its high efficacy and simplicity. Genome editing tools have also been established in silkworm (Bombyx mori), a model organism of Lepidoptera insects with high economic importance. This has remarkably improved the level and scope of silkworm research and could reveal new mechanisms or targets in basic entomology and pest management studies. In this review, we summarize the progress and potential of genome editing in silkworm and its applications in functional genomic studies for generating novel genetic materials.

Identification and characterization of toxins in the venom gland of the Chinese bird spider, Haplopelma hainanum, by transcriptomic analysis

Tarantula venoms provide a model system for studying toxin selectivity, structure-activity relationships and molecular evolution ofpeptide toxins. Previous studies have identified a large number of peptide toxins in the venom of the Chinese bird spider Haplopelma hainanum, generally regarded as a highly venomous spider. However, the lack of available RNA-seq transcriptomic and genomic data is an obstacle to understanding its venom at the molecular level. In this study, we investigated the venom gland transcriptome of/-/, hainanum by RNA-seq, in the absence of an available genomic sequence. We identi- fied 201 potential toxins among 57 18 l de novo assembled transcripts, including knottins, Kunitz-type toxins, enzymes and other proteins. We systematically identified most of the knottins and Kunitz-type toxins, some of which showed strongly biased expression in the venom gland, including members of the huwentoxin-1, huwentoxin-2 and magi-1 families. We also discovered several novel potential toxins. These data demonstrate the high molec- ular and structural diversity in the venom toxins ofH. hainanum. This study offers a useful strategy for exploring the complex components of spider venoms.

Characterization of the Bombyx mori Cecropin A 1 promoter regulated by IMD pathway

Cecropin A1 （CeeA1） promoter from Bombyx mori was cloned and character- ized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecAl. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other pre- dicted elements on CecAl promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecAl promoter. Furthermore, binding of Relish to lob motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecAl expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications.

Genome-wide identification of chitin-binding proteins and characterization of BmCBP1 in the silkworm, Bombyx mori

The insect cuticle plays important roles in numerous physiological functions to protect the body from invasion of pathogens, physical injury and dehydration. In this report, we conducted a comprehensive genome-wide search for genes encoding proteins with peritrophin A-type (ChtBD2) chitin-binding domain (CBD) in the silkworm, Bombyx mori. One of these genes, which encodes the cuticle protein BmCBP1, was additionally cloned, and its expression and location during the process of development and molting in B. mori were investigated. In total, 46 protein-coding genes were identified in the silkworm genome, including those encoding 15 cuticle proteins analogous to peritrophins with one CBD (CPAP1s), nine cuticle proteins analogous to peritrophins with three CBD (CPAP3s), 15 peritrophic membrane proteins (PMPs), four chitinases, and three chitin deacetylases, which contained at least one ChtBD2 domain. Microarray analysis indicated that CPAP-encoding genes were widely expressed in various tissues, whereas PMP genes were highly expressed in the midgut. Quantitative polymerase chain reaction and western blotting showed that the cuticle protein BmCBP1 was highly expressed in the epidermis and head, particularly during molting and metamorphosis. An immunofluorescence study revealed that chitin co-localized with BmCBP1 at the epidermal surface during molting. Additionally, BmCBP1 was notably up-regulated by 20-hydroxyecdysone treatment. These results provide a genome-level view of the chitin-binding protein in silkworm and suggest that BmCBP1 participates in the formation of the new cuticle during molting.

Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpfl) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP 16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.

Genome-wide identification of target genes for transcription factor BR-C in the silkworm,Bombyx mori

Transcription factor Broad Complex(BR-C)is an ecdysone primary response gene in insects and participates in the regulation of insect growth and development.In this study,we performed a genome-wide identification of BR-C target genes in silkworm(Bombyx mori)using chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-seq).As a result,a total of 1006 BR-C ChIP peaks were identified,and 15%of peaks were located in the promoter regions of 133 protein-coding genes.Functional annotation revealed that these ChIP peak-associated genes,as potential BR-C targets,were enriched in pathways related to biosynthetic process,metabolic process,and development.Transcriptome analysis and quantitative real-time polymerase chain reaction(PCR)examination revealed that developmental changes in expression patterns of a portion of potential BR-C targets,including HR96 and GC-otl,were similar to those of BR-C.ChIP-PCR examination confirmed that BR-C could directly bind to the promoters of potential targets.Further,dual luciferase assays demonstrated that HR96 promoter activity was significantly upregulated following BR-C overexpression,and this upregu-lation was abolished when the binding motif in the promoter was truncated.This study will be helpful for deciphering the regulatory roles of BR-C during insect growth and development.

Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection

Dear Editor, The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has become a popular and powerful method for manipulating genomes with considerably little effort and high efficiency. It allows for the generation of targeted indels, genomic structure variations, and insertion of foreign fragments. However, increasing concerns have been raised about possible off-target effects.

The silkworm homolog of Methoprene-tolerant （Met） gene reveals sequence conservation but function divergence

Methoprene-tolerant （Met） gene has been found to be involved in juvenile hormone （JH） action in insects. Herein, we isolated a silkworm （Bombyx mori） homolog of Met gene from Drosophila melanogaster using bio-informatics analysis and rapid amplification of cDNA ends - polymerase chain reaction method, and defined it as BmMet. The full-length cDNA ofBmMet gene consists of 1 917 nucleotides and includes a 1 368 bp of open reading frame for a deduced protein of 455 amino acids. All deduced protein sequences from Met genes in B. mori and other surveyed insects contain four typical domains ofbHLH, PAS-A, PAS-B and PAC, highlighting a high sequence conservation of Met genes during insect evolution. Also, genomic structure and phylogenic analysis suggested that Met in both B. mori and Drosophila species may originate from an ancestor gene with gce, another member of bHLH-PAS family, via gene duplication. In addition, BmMet was detected in all surveyed tissues and throughout the whole life of silkworm at transcriptional levels. Furthermore, silkworm individuals with RNAi silencing of BmMet gene in the early stage of the fourth instar larvae could molt normally and pupate successfully. This result was different from the observation in T. castaneum but similar to that in D. melanogaster after Met knockdown, revealing that the action mode of Met in B. mori and D. melanogaster should be divergent with that in other insect species.

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era.Streptomyces phage-derived phiC31 integrase,which mediates an irreversible site-specific cassette exchange between the phage attachment site(attP)and the bacterial attachment site(attB),provides a promising option for the construction of a controllable gene-expression system.Here,we report a phiC3I integrase-mediated promoter flip system(FLIP)for the inducible expression of target genes in silk-worm(Bombyx mori).First,we constructed a FLIP reporter system,in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene.The coexpression of a C-terminal modified phiC3 I-NLS integrase carrying a simian virus 40(SV40)nuclear localization signal(NLS)effectively flipped the BmAct4 promoter through an attBlattP exchange,thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line,BmE.Subsequently,the FLIP system,together with a system continuously expressing the phiC3 I-NLS integrase,was used to construct binary transgenic silkworm lines.Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter,with an approximately 39%heritable transformation efficiency in silkworm offispring,leading to the constitutive and high-level expression of DsRed in silkworms,which accounted for approximately 0.81%of the silkworm pupal weight.Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.

Genetically engineered FGF1-sericin hydrogel material treats intrauterine adhesion and restores fertility in rat

Endometrial injury can cause intrauterine adhesions(IUA)and induce the formation of endometrial fibrosis,leading to infertility and miscarriage.At present,there is no effective treatment method for severe IUA and uterine basal injury with adhesion area larger than one-third of the uterus.In this study,we prepared FGF1 silk sericin hydrogel material(FGF1-SS hydrogel)to treat endometrial injury and prevent endometrial fibrosis.Compared with the silk sericin hydrogel material(WT-SS hydrogel),FGF1-SS hydrogel significantly promotes the cell migration and infiltration ability of endometrial stromal cells(ESCs).More importantly,FGF1-SS hydrogel can release FGF1 stably for a long time and inhibit the ESCs injury model forms fibrosis through the TGF-β/Smad pathway.In the IUA rat model,FGF1-SS hydrogel treatment effectively restored the number of uterine glands and uterine wall thickness in rats,with a fertility rate of 65.1%66.4%.The results show that FGF1-SS hydrogel is expected to be a candidate to prevent IUA.

CRISPR/dCas9-mediated imaging of endogenous genomic loci in living Bombyx mori cells

Dear Editor,Visualization of specific endogenous genomic loci is crucial to understand the molecular basis underlying the spatiotemporal regulation of gene expression.Specific genetic loci were previously visualized primarily via fluorescent in situ hybridization(FISH)in fixed cells(Ma et al.,2016).

Targeted activation of BmCyclinE in Bombyx mori using designer TAL effectors

Dear Editor, Artificial expression systems are crucial to functional genomics studies, constructing synthetic genetic circuits, and bioengineering (Werner & Gossen, 2014). They are usually achieved by cloning the coding sequence (CDS) of a target gene into a vector containing natural or engineered regulatory elements. However, this strategy is limited when target genes are hard to clone, for example when the CDS is too long, or expression level is too low.

Identification of MBF2 family genes in Bombyx mori and their expression in different tissues and stages and in response to Bacillus bombysepticus infection and starvation

The Multiprotein bridge factor 2 （MBF2） gene was first identified as a coactivator involved in BmFTZ-F 1-mediated activation of the Fushi tarazu gene. Herein, nine homologous genes of MBF2 gene are identified. Evolutionary analysis showed that this gene family is insect-specific and that the family members are closely related to response to pathogens （REPAT） genes. Tissue distribution analysis revealed that these genes could be expressed in a tissue-specific manner. Developmental profiles analysis showed that the MBF2 gene family members were highly expressed in the different stages. Analysis of the expression patterns of nine MBF2 family genes showed that Bacillus bombysepticus treatment induced the up-regulation of several MBF2 family genes, including MBF2-4, -7, -9, -8. Furthermore, we found the MBF2 family genes were modulated by starvation and the expression of these genes recovered upon re-feeding, except for MBF2-5, -9. These findings suggested roles for these proteins in insect defense against pathogens and nutrient metabolism, which has an important guiding significance for designing pest control strategies.